A cell-free protein-synthesizing system active in initiation of translation of both endogenous mRNA and exogenous mRNA has been obtained from postmitochondrial supernatant (S-12) of the liver of ethionine-treated rats by adding reticulocyte ribosomal extract as a source of initiation factor. Formation of polysomes in the course of protein synthesis in vitro has also been demonstrated. Homogenization of the liver in the presence of 50 μM hemin stabilizes the initiation activity of S-12 fraction, which otherwise decays rapidly even at 0&C. The mechanism of inhibition of protein synthesis by ethionine is discussed in view of these results.

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Soluble NADH-cytochrome b₅ reductase was purified from rabbit erythrocytes to homogeneity by simple procedures developed in this study including fractionation with ammonium sulfate, gel filtration on a Sephadex G-75 column, and affinity chromatography on a 5'-AMP-Sepharose 4 B column. The enzyme was purified about 12, 000-fold from hemolysate in terms of NADH-cytochrome b₅ reductase activity with a high yield of 40%. The purified enzyme has absorption maxima at 273, 390, and 462 nm, and shoulders at 370, 435, and 488 nm. The ratio of the absorbance at 273 nm to that at 462 nm of the purified enzyme was 5.6-5.8. The prosthetic group of the enzyme was found to be FAD, and the flavin content in the enzyme was 1 mol/mol of the enzyme. The molecular weight of the purified enzyme was estimated to be 33, 000 and 32, 000 by gel filtration on a Sephadex G-75 column, and by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, respectively. The NADH-cytochrome b₅ reductase activity decreased strikingly as the buffer or salt concentration in the assay mixture was increased, and the optimal pH for the reduction of cytochrome b₅ with NADH was determined to be 6.6 in Tris-maleate buffer of constant ionic strength. The maximum velocity of NADH-cytochrome b₅ reductase activity of the purified enzyme was very high, 1, 280 μmol/min/mg of protein in 10mM phosphate buffer (pH 6.6). The Michaelis constants for NADH and cytochrome b₅ were determined to be 2.5 and 4 μm, respectively. The reduction of cytochrome b₅ with NADH by the enzyme was suggested to follow the ordered-type reaction mechanism based on the modes of product inhibition. From these results, and also from the estimated enzyme content in the erythrocytes (16-20mg protein per liter of packed erythrocytes), the possible contribution of the enzyme to functions other than methemoglobin reduction in rabbit erythrocytes is discussed.

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Biliverdins formed from heme by a microsomal preparation and a reconstituted heme oxygenase system were each converted to their dimethyl esters and analyzed for isomeric composition by reversed-phase high-performance liquid chromatography, using a column of μBondapak C₁₈ (Waters Associates); on this column, the dimethyl esters of four biliverdin IX isomers, that is IXα, IXβ, IXγ, and IXδ, have been shown to be eluted separately in the order IXα, IXβ, IXδ, and IXγ, when developed with methanol/water. The analysis indicated that the enzymatically formed biliverdins were exclusively IXα; the elution profile exhibited no other significant elution peak due to other biliverdin isomers. It was concluded that the heme oxygenase system cleaves the heme ring specifically at the α-methene bridge to yield biliverdin IXα.

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The administration to rats of leupeptin produced prominent numbers of enlarged and irregularly shaped autolysosomes in hepatocytes. Percoll density equilibration of crude lysosomal fractions from rat livers showed that most lysosomal enzyme migrated from normal lysosomal density fractions toward higher density fractions within 30min after leupeptin injection. The denser particles were ultrastructurally identified with the autolysosomes. In analysis by differential centrifugation of the liver homogenates, lysosomal enzymes became sedimentable with particles of greater sedimentation rate within 30min after leupeptin injection. These changes in physical properties of lysosomes reverted to normal after 24 h, and concomitant disappearance of autolysosomes in hepatocytes was observed by electron microscopy. When the time course of distribution of leupeptin in subcellular fractions was analyzed, particle-bound leupeptin shifted with time to larger particle fractions in parallel with the shift of lysosomal enzyme distribution. Upon injection with leupeptin, cathepsin B activity was inhibited by more than 80% for about 3 h and was gradually restored to a normal level after 24 h. Leupeptin treatment caused marked delay of the degradation of endocytosed FITC-labeled asialofetuin in hepatic lysosomes, and its inhibitory effect lasted for over 9 h after the injection. It was suggested that autophagy is probably a normal process of protein degradation in hepatocytes, and because of a retarded digestion of sequestered materials, autolysosomes persisted for a long period and made up the majority of the lysosome population in the leupeptin-treated cells.

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1. RNase Ms, a base non-specific RNase from Aspergillus saitoi was reduced and carboxymethylated (RCM-RNase Ms). RCM-RNase Ms was hydrolyzed with trypsin, and the trypsin digests were then treated with chymotrypsin. Trypsin digests were also treated with Staphylococcal protease and with chymotrypsin, separately.
2. By the analyses of the amino acid sequences of the peptides formed, the alignment of these peptides in RCM-RNase Ms was determined.
3. From the digest of heat-denatured RNase Ms with Bacillus subtilis protease, two peptides containing disulfide bridges were isolated. From the analyses of these two peptides, the locations of the bridges were determined.
4. The amino acid sequence of RNase Ms was compared with those of RNase T₁ (Asp. oryzae, guanine specific), RNase U₁ (Ustilago sphaerogena, guanine specific) and RNase U₂ (Ustilago sphaerogena, purine specific). There are very similar sequences between these four RNases irrespective of their differences in base specificity. These were, in RNase Ms, tripeptide sequence containing His₃₉ (Tyr-Pro-His), the tetrapeptide containing Glu₅₇ (Glu-Tyr-Pro-Ile), the hexapeptide containing Arg₇₆ (Asp-Arg-Val-Ile-Phe-Asp) and the hexapeptide containing His₉₁ (Ile-Thr-His-Thr-Gly-Ala). The other sequences common for all four RNases are Tyr₆₇, Pheloo, and Cys₁₀₃ in RNase Ms. Since among these peptides His₃₉, Glub₇, His₉₁, and Arg₇₆ in RNase Ms corresponded to His₄₀, Glu₅₈, His₉₂, and Arg₇₇ in RNase T₁ which are known to be involved in the active site of RNase T₁, the possible role of these amino acids in the active site of RNase Ms is discussed.
5. The sequence similarity of RNase Ms to that of RNase T₁ was about 60% and to those of RNase U₁ and RNase U₂ was about 30%.
6. The details of the experimental evidence used to elucidate the amino acid sequence of RNase Ms are described in the supplemental miniprint.

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The turnover rates of mitochondrial and cytosolic fumarase in the rat liver were determined by injecting L-[U-¹⁴C] leucine and following the decay of specific radioactivity incorporated into immunoprecipitates from the partially purified enzymes. The half-life of mitochondrial fumarase (t_1/2=9.7 days) was significantly different from that of the cytosolic enzyme (t_1/2=4.8 days). Studies on the incorporation of radioactive leucine into fumarase in the liver under steady-state conditions showed that the rate of synthesis of this enzyme in cytosol was about 2 times higher than that in the mitochondrial enzyme. The results showed that the mitochondria) fumarase turns over considerably more slowly than the cytosolic enzyme in the rat liver. These results suggest that the turnover of two fumarases with different localizations may be under different and independent control systems.
In the case of the mitochondrial fumarase, the decay curve of its specific radioactivity obtained by single injection of L-[U-¹⁴C] leucine was quite unusual. No change was observed in the specific radioactivity of the mitochondrial fumarase for about 7 days after pulse labeling, then the specific radioactivity decreased exponentially with a half-life of 9.7 days.

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β₂-Bungarotoxin (β₂-toxin) was isolated from the venom of Bungarus multicinctus by means of CM-Sephadex C-25 column chromatography, Sephadex G-75 gel filtration and CM-Sephadex C-25 column rechromatography. β₂-Toxin consisted of two dissimilar polypeptides, A (120 amino acid residues) and B (60 amino acid residues) chains, crosslinked by an interchain disulfide bond. The neurotoxicity (LD₅₀) and phospholipase activity of β₂-toxin were 0.029 μg/g of mouse and 48.9 units/mg of toxin, respectively, and both the activities were slightly weaker than those (0.019 μg/g and 60.9 units/mg) of β₁-bungarotoxin (β₁-toxin).
β₂-Toxin was reduced and carboxymethylated and then its RCM-A and -B chains were separated. Each RCM-chain was maleylated and then digested with TPCK-trypsin. The tryptic peptides were sequenced by manual Edman degradation or the dansyl-Edman method, and the total alignment of the tryptic peptides from each RCM-chain was deduced based on the amino acid sequences of the A and B chains of β₁-toxin. The amino acid sequence of the B chain of β₂-toxin differed from that of the B chain of β₁ toxin by 22 amino acid substitutions, while those of their A chains were identical. We concluded that the variation in the amino acid sequence of the B chains did not significantly affect the neurotoxicity of the β-toxins.
The amino acid sequences of the B chains of the two β-toxins were homologous to those of proteinase inhibitors from snake venoms and mammalian pancreas, but no inhibitory activity of the two β-toxins on proteinases was observed.

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The two most basic β-bungarotoxins (β3- and β₄-toxins) and another, less neurotoxic β-bungarotoxin (β₅ toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD₅₀ values of the β₃ and β₄-toxins were 0.066 μg and 0.072 μg/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. β₅-Toxin was weaker in neurotoxicity (LD₅₀, 0.13 μg/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin.
Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of β₁-bungarotoxin (β₁-toxin). The amino acid sequences of the A chains of the β₃- and β₄-toxins were identical but differed from those of the A chains of the β₁- and β₂-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the β₃ and β₄-toxins differed from each other, but they were identical with those of the B chains of the β₁- and β₁-toxins, respectively. The amino acid sequence of the A chain of β₅ toxin differed from that of the A chain of β₁-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of β₅-toxin was identical with those of the B chains of β₁ and β₃ toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the β-toxins.

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Metapyrocatechase [catechol: oxygen 2, 3-oxidoreductase, EC 1. 13. 11. 2] was immobilized with cyanogen bromide-activated agarose with a coupling yield of 78% of the protein added. The immobilized enzyme showed a specific activity of 77 μmol/min/mg protein, which is about 30% of that of the native enzyme. The immobilization enhanced the stability of the enzyme against inactivation by heat, acid or alkaline pH, and various denaturing agents. However, like the native enzyme, the immobilized enzyme was rapidly inactivated by oxidants such as oxygen or hydrogen peroxide, and the inactivation was completely prevented in the presence of low concentrations of organic solvents. The pH profile for the activity and the substrate specificity were slightly changed by the immobilization.

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Glucose oxidase from Aspergillus niger, hyaluronidase from Streptomyces hyalurolyticus, and cholesterol oxidase and cholesterol esterase from Pseudomonas fluorescens were effectively adsorbed on an Amberlite CG-50 column, when the cell-free cultured medium or the cultured medium with cell extract and without cell debris was applied without desalting but at pH≤4.5. At the acidic pH, all the ion-exchange groups (-COOH) exist in the protonated form; the adsorption is not due to electrostatic attraction, but to hydrophobic interaction. The enzymes thus adsorbed were effectively eluted by increasing pH, at which the ion-exchange groups became dissociated. This type of adsorption-elution is called hydrophobic-ionic chromatography. By a single run of chromatography, glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase were purified 30-fold, 12-fold, 45-fold, and 20-fold with yields of 82%, 83%, 80%, and 90%, respectively. This indicates that hydrophobicionic chromatography on an Amberlite CG-50 column is effective for the purification of various enzymes, provided that they are stable at the acidic pH.

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Conditions for in vitro assembly and disassembly of Tetrahymena 14-nm filaments were investigated electron-microscopically by using a crude extract of acetone powder of the cells. The assembly conditions established are: incubation of a protein sample (2mg/ml) in 5mM 2-(N-morpholino) ethanesulfonic aicd (MES) buffer (pH 6.6) containing 0.1mM N^α-tosyl-L-lysyl-chloromethane hydrochloride (TLCK), 50mM KCl, 0.6mM ATP, and 1.2mM CaCl₂ at 30&C for 30min. The disassembly conditions established are: dialysis of the 14-nm filament suspension (3mg protein/ml) against Tris-acetate buffer (pH 8.2) containing 5mM 2-mercaptoethanol, 1mM ethylene glycol bis(β-aminoethyl ether)-N, N'-tetraacetic acid (EGTA), and 0.05mM TLCK at 4&C for 24 h. The assembly and disassembly were repeatable, and resulted in the exclusive retention of the 49, 000-dalton protein. This clearly shows that the previously reported protein component (38, 000-dalton protein: FFP-38) of the 14-nm filament is incorrect and the actual component is indeed a 49, 000-dalton protein.
The present research also showed that the Tetrahymena 14-nm filament bore a strong resemblance to ‘intermediate filaments’ of mammalian cells with respect to molecular weight, amino-acid composition of the protein component, and size and conditions for assembly and disassembly of the filament.

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We have applied the fluorescent probes 1-anilino-8-naphthalene sulfonate (ANS), and pyrene, and an SH-directed reagent, 5, 5'-dithiobis (2-nitrobenzoate) (DTNB), in order to analyze Ca²⁺-induced conformational changes in porcine intestinal brush border membranes. Addition of Ca²⁺ to the ANS-membrane complex resulted in enhancement of the fluorescence intensity accompanied by an increase of the affinity of the dye for the membranes, shift of the transition temperature of the ANS fluorescence toward a higher temperature range, and disappearance of the slope in the thermal Perrin plot of the fluorescence polarization. These results suggest that the membrane structure becomes more rigid upon binding of Ca²⁺ to the membranes. This hypothesis was further confirmed by a marked decrease of the excimer formation efficiency of pyrene in the membranes with increasing Ca²⁺ concentration. Reaction of the membranes with DTNB suggested that there are at least two classes of SH groups with different pseudo-first-order rate constants, 1.48×10^-3_S^-1 (SH_f) and 0.25×10^-3_S (SH_s) at pH 7.4 in the absence of Ca₂₊. Of the estimated 52.2 nmol of SH groups per mg protein in the presence of sodium dodecyl sulfate, about 23.8 nmol are reactive with DTNB under the same conditions. The reactivity of SH groups with DTNB is responsive to Ca²⁺ concentration and the maximum number of reactive SH groups was increased by the addition of Ca₂₊.
Based on these results, the conformational changes of the membrane structure induced by Ca²⁺ are discussed.

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As a continuation of our work on toxin A-chain conjugates with antitumor antibodies for selective delivery of the toxin to the target cells, four ricin A-chain conjugates were prepared by linking A-chain to Fab' or F (ab')₂ of rabbit IgG against L 1210 with or without employing a cross-linking agent, N, N'-o-phenylenedimaleimide (PDM), N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) or N-succinimidyl m-(N-maleimido) benzoate (SMB), and the effects of antigen-binding valency and of the nature of the cross-linking bond on their in vitro cytotoxicity were studied. The relative potencies of the conjugates in terms of IC₉₄'s were as follows: F (ab')₂-SPDP-A-chain, 100; Fab'-S-S-A-chain, 21; F (ab')₂-SMB-A-chain, 1.3; Fab'-PDM-A-chain 0.38. Among the four conjugates, F (ab')₂-SPDP-A-chain and Fab'-S-S-A-chain can be cleaved into the homing and the cytotoxic components with 2mM 2-mercaptoethanol. These results suggest that divalency in antigen-binding and susceptibility of the cross-linking bond to cleavage by mercapto reagent are desirable for high potency. Protein synthesis in a cell-free system of rabbit reticulocyte lysate was inhibited by Fab'-S-S-A-chain and by Fab'-PDM-A-chain as effectively as by free A-chain, indicating that the liberation of A-chain is not important, at least on ribosomes, but it is important for the A-chain to reach a ribosome after binding of the conjugates to the cell-surface.

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Nearly full-sized double-stranded cDNA was prepared from virion RNA of poliovirus Sabin 1 (LSc, 2 ab) strain using reverse transcriptase. The double-stranded cDNA was cleaved at four and two sites by the restriction endonucleases Bam HI and Hind III, respectively. Based on the cleavage patterns of double-stranded cDNA into segments of various lengths, each of which had the sequence corresponding to that of the 3'-end of the viral genome, the location of each restriction fragment was determined. The cDNA fragments were cloned with pBR 322 as a vector and some of their nucleotide sequences were determined. The DNA sequence indicated that the restriction map obtained was consistent with the known arrangement of viral RNA fragments (1-3). Comparison of the nucleotide sequences of the cloned Hind III (460 bases) and Pst I (434 bases) fragments with those of the corresponding region of Mahoney type 1 genome (3) revealed 12 point-mutation sites.

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Precise immunological recognition of anti-viomycin antiserum at detailed parts in the structure of viomycin was studied by cross reactivities of the antiserum to viomycin and its ten analogs using an enzyme immunoassay of viomycin. The antiserum clearly recognized all minor modifications in the sixteen membered ring of viomycin, indicating that the antiserum clearly recognizes the whole structure of the sixteen membered ring. Recognition of the antiserum on the α-lysine terminus was also examined showing that the antiserum was also recognized on this part. Thus, the anti-viomycin antiserum was deduced to recognize the whole structure of viomycin, from which the deduction was made that the anti-viomycin antibodies in the antiserum must possess cavities fitting the whole structure of viomycin. The crystal dimensions of viomycin are 13 Å in length, 8 Å in width, and 7 Å in depth. Thus, the high dimensional structure of the binding sites of the anti-viomycin antibodies was deduced to possess cavities of a similar size to that of viomycin.

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The effect of various concentrations of potassium salts on the activity of the N-ethylmaleimide (NEM)-treated hemin-controlled translational inhibitor (HCI) was investigated using the rabbit reticulocyte lysate system.
The NEM-treated HCI was found to be less active for the inhibition of globin synthesis at higher concentrations of potassium salts. In addition, the binding of [³⁵S] Met-tRNA_f to the 40 S ribosomal subunits was inhibited more profoundly at 100mM than at 240mM KOAc.
The phosphorylation of the NEM-treated HCI and eukaryotic initiation factor, eIF-2, was studied at various concentrations of KOAc. The NEM-treated HCI was phosphorylated to an almost constant extent at 20 to 300mM K⁺, but the phosphorylation of eIF-2α by the inhibitor decreased with increasing concentration of KOAc. It was also found that the incubation of the ³²P-labeled inhibitor with eIF-2 did not produce the ³²P-labeled eIF-2.
In the light of these data, a possible relationship between the phosphorylation of the inhibitor or eIF-2α and the inhibition of globin synthesis by the inhibitor is discussed.

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Translation of poly (A) in eukaryotic cell-free systems was investigated in comparison with that of poly (U), using the E. coli cell-free system as a control, and the following results were obtained.
1. Poly (A) was not translated in the eukaryotic cell-free systems prepared from yeast, wheat embryos, and rat liver, whereas poly (U) was well translated in these systems. On the other hand, fairly good translation of poly (A) as well as poly (U) was observed in the E. coli cell-free system.
2. The inefficient translation of poly (A) observed in the eukaryotic systems was not due to the lack of lysyl-tRNA synthetase, because the addition of [¹⁴C] lysyl-tRNA did not improve the synthesis of polylysine dependent on poly (A).
3. Eukaryotic ribosomes such as those prepared from Artemia salina did not bind [³H] poly (A) but bound [³H] poly (U) in the absence of tRNA, whereas they bound both polynucleotides if tRNA was present. Furthermore, the binding of [³H] poly (A) to A. salina ribosomes observed in the presence of tRNA was strongly dependent on the incubation temperature, indicating that the interaction between ribosomes and poly (A) was hampered by the base stacking known to be present in poly (A). Thus, tRNA seemed to be required for the formation of the stable ribosome-poly (A) complex.
4. Binding of [¹⁴C] lysyl-tRNA to ribosomes, either of A. salina or of E. coli, was observed as well as that of [¹⁴C] phenylalanyl-tRNA. In contrast, chain elongation of polylysine was extremely slow in the eukaryotic system as compared with the E. coli system, although chain elongation of polyphenylalanine proceeded equally well in both systems, and this resulted in poor synthesis of polylysine in the eukaryotic systems.

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Interaction of adenosine-5'-O-(3-thiotriphosphate) (ATPγS) with Ca²⁺, Mg²⁺-ATPase of sarcoplasmic reticulum was studied. The nucleotide was slowly hydrolyzed by the ATPase at 30&C at a rate of about 0.5% that of ATP hydrolysis. Whereas at 0&C, ATPγS showed only a limited reactivity toward the ATPase in that a thiophosphorylated intermediate was formed and ADP was released, but hydrolysis of the intermediate to complete the catalytic cycle did not occur. A fairly stable analog of the E-P intermediate could thus be obtained. Presence of the thiophosphorylated intermediate was indicated by the [³H] ADPE↔ATPγS exchange reaction and also by using [³⁵S] ATPγS. When the ATPase was reacted with ATPγS at 0&C in the presence of ferricyanide, EP-forming activity was rapidly lost. Free Ca²⁺ ions were required for this inactivation. Disulfide bond formation between a cysteinyl residue located near the substrate binding site and the enzyme-bound ATPγS or the thiophosphorylated intermediate was suggested by the fact that 2-mercapto-ethanol reversed the inactivation. The reaction may prove to be a useful tool for affinity labeling of the active site of the ATPase.

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1. The addition of palmitic acid or myristic acid to a medium containing delipidated fetal bovine serum resulted in severe inhibition of V 79-R cell growth. The degree of inhibition by palmitic acid was concentration dependent. Simultaneous addition of oleic acid protected the cells from the inhibition by palmitic acid.
2. In the presence of palmitic acid, total phospholipid and triacylglycerol per cell increased to 2- and 13-fold, respectively.
3. Palmitic acid caused an increase in the proportion of palmitic acid with concomitant decrease of oleic acid in phosphatidylcholine, phosphatidylethanolamine and triacylglycerol.
4. Palmitic acid inhibited the synthesis of phospholipid molecular species with two monoenoic fatty acids. However, membrane flow of phospholipids from endoplasmic reticulum to plasma membrane was preserved.
5. About 70% of the triacylglycerol molecular species were those containing three saturated or two saturated and one monoenoic fatty acids.
6. Electron microscopy revealed a large amount of triacylglycerol and fiber-like membrane structures in the cells supplemented with palmitic acid.

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The alpha band of the native ferric low-spin form of cytochrome P-450 is more intense than the β band and this is characteristic of the cytochrome. When the native sixth ligand of P-450₁, a low-spin cytochrome P-450 of rabbit liver microsomes, was replaced with an exogenous ligand, the α band diminished and the cytochrome lost the above-mentioned spectral characteristics. This indicated that the intense a band of the cytochrome was related to the nature of its sixth ligand.
P-448₁, a rabbit liver microsomal cytochrome P-450 which is essentially in a high-spin state, was converted to low-spin complexes upon binding with various exogenous ligands. The intensity of the α band of low-spin P-448₁ derivatives increased in the order of 1-methylimidazole and pyridine complexes, 2-methylimidazole and 2-methylpyridine complexes, and lower n-alkanol complexes. This order was coincident with the decreasing order of field strength of the exogenous ligands, indicating that the intensity of the α band is inversely proportional to the field strength of the sixth ligand.
The absorption spectrum of the 1-propanol complex of P-448₁ was essentially identical with that of native low-spin cytochrome P-450, whereas spectra of other P-448₁ derivatives were superimposable on those of the corresponding P-450₁ derivatives.
P-450₁ and metmyoglobin showed an orderly red-shift of the Soret band upon binding with pyridine, 1-methylimidazole and cyanide. The order and the degree of the red-shift of the Soret band of P-450₁ were essentially the same as those of metmyoglobin, indicating that the native sixth ligand of P-450₁ may resemble that of metmyoglobin, a water molecule.
From these observations, we propose that the native sixth ligand of ferric lowspin cytochrome P-450 is an oxygenous one such as a water molecule or an oxyamino acid side chain from the apoprotein.

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1. In order to elucidate the structure-function relation of a glucoamylase [EC 3. 2. 1. 3, α-D-(1→4) glucan glucohydrolase] from Aspergillus saitoi (Gluc M₁), the reaction of Gluc M₁ with NBS was studied.
2. The tryptophan residues in Gluc M₁ were oxidized at various NBS/Gluc M₁ ratios. The enzymatic activity decreased to about 80% of that of the native Gluc M₁ with the oxidation of the first 2 tryptophan residues. The oxidation of these 2 tryptophan residues occurred within 0.2-0.5 s. On further oxidation of ca. 4-5 more tryptophan residues of Gluc M₁, the enzymatic activity of Gluc M₁ decreased to almost zero (NBS/Gluc M₁=20). Thus, the most essential tryptophan residue (s) is amongst these 4-5 tryptophan residues.
3. 7.5 tryptophan residues were found to be eventually oxidized with increasing concentrations of NBS up to NBS/Gluc M₁=50. This value is comparable to the number of tryptophan residues which are located on the surface of the enzyme as judged from the solvent perturbation difference spectrum with ethylene glycol as perturbant.
4. In the presence of 10%. soluble starch, about 5 tryptophan residues in Gluc M₁ were oxidized at an NBS/Gluc M₁ ratio of 20. The remaining activity of Gluc M₁ at this stage of oxidation was about 76%. On further oxidation, after removal of soluble starch, the enzymatic activity decreased to zero with the concomitant oxidation of 2 tryptophan residues. The results indicated that the essential tryptophan residue (s) is amongst these 2 tryptophans.
5. The UV difference spectrum induced by addition of maltose and maltitol to Gluc M₁ showed 4 troughs at 281, 289, 297, and 303 nm. The latter 3 troughs were probably due to tryptophan residues of Gluc M₁ and decreased with NBS oxidation.

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1. Porcine cardiac native tropomyosin was phosphorylated by bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Most of the phosphate incorporation was observed in troponin I, the maximum of which was 0.7 mol of P₁ per mol of troponin I.
2. In the presence of phosphorylated native tropomyosin, actomyosin ATPase activity was 15-40% lower than that in the presence of the unphosphorylated preparation at all calcium ion concentrations (1.5×10^-8M-2.4×10^-5M). Half-maximum activation of ATPase was obtained with a concentration of 7×10^-7M Ca²⁺ (unphosphorylated) and 1.3×10^-6M Ca²⁺ (phosphorylated), respectively. Maximum ATPase activity was reached with 3×10^-6M Ca₂₊ (unphosphorylated) and 1.0×10^-6M Ca²⁺, (phosphorylated).
3. Porcine cardiac troponin I isolated by affinity chromatography inhibited ATPase activity of desensitized actomyosin in the presence of tropomyosin. There was little difference between phosphorylated troponin I and a control preparation with regard to the inhibitory effect on ATPase activity.
4. Troponin C from rabbit skeletal muscle neutralized the inhibitory effect of troponin I. The minimum amount of troponin C required for complete neutralization was approximately equimolar to troponin I. The inhibitory effect of phosphorylated troponin I was neutralized by troponin C less effectively than that of unphosphorylated preparation.

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Two radioiodination methods for surface proteins on mouse lymphocyte membrane, that is, the lactoperoxidase method and the chloramine T method, were comparatively investigated. The extents of iodination under various conditions were determined by measurements of radioactivities incorporated into the cell fractions and into the protein components isolated by specific immunoprecipitation from lyzates of the labeled cells. It was demonstrated that the chloramine T method, as well as the lactoperoxidase method, was applicable to label the lymphocyte surface proteins without any significant labeling of the cytoplasmic proteins. It was also found that the iodination efficiencies of the membrane immunoglobulin polypeptide chains in these two labeling methods were different in each of the isotypes, μ and δ chains. The possibility that the differences are related to their primary structures and to their exposure or anchorage profiles on the cell membrane is discussed with reference to the reaction mechanisms of the two labeling methods.

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Previously, we (Onishi et al. 1978, Suzuki et al. 1978, and Suzuki et al. 1981) reported studies on gizzard unphosphorylated myosin (UM) in MgCl₂ concentrations lower than 10mM. In the present report, we describe some findings of a study of UM in MgCl₂ concentrations higher than 10mM.
1. The turbidity of UM suspensions in the presence of 1mM ATP increased as the MgCl₂ concentration increased to higher than 10mM, suggesting that UM can form “ATP-resistant” filaments in such extremely high concentrations of MgCl₂. This suggestion was supported by electron microscopic observation.
2. The ATPase activity of UM alone also increased as the MgCl₂ concentration increased, indicating that the activity is much higher with UM in a filamentous form than with UM in a disassembled form.
3. As the MgCl₂ concentration increased to higher than 10mM, both actin-activation of UM-ATPase and ATP-induced superprecipitation occurred with increasing magnitude.
Summarizing these results, we observed that the requirement of MgCl₂ in a concentration higher than 10mM was common for formation of ATP-resistant filaments from UM, for increase in the ATPase activity of UM, and for superprecipitation of acto-UM. Moreover, since these findings for UM are very similar to those obtained previously (Suzuki et al. 1978, and 1981) with gizzard phosphorylated myosin (PM), except that MgCl₂ in a concentration higher than 2mM was the common requirement for PM, we propose a) that formation of ATP-resistant filaments from myosin is the cause of the increase in the ATPase activity of myosin alone and for activation of ATP-induced superprecipitation, and b) that the primary effect of phosphorylation of gizzard myosin light chains is on the MgCl₂ concentration required for formation of ATP-resistant filaments of myosin, reducing it (>10mM) to the physiological concentration (>2mM).
4. In connection with the latter proposal, we observed, by measuring the turbidity of myosin suspensions, that Ca²⁺, Sr²⁺, and Mn²⁺ can be used in place of Mg²⁺ to form ATP-resistant filaments of UM or PM, and that the concentration of these divalent cations required for formation of ATP-resistant filaments was again lower with PM than with UM.
5. We also measured the sedimentation rate and the ATPase activity of both UM and PM in the presence of MgCl₂ where neither UM nor PM formed ATP-resistant filaments. We thus observed (in the presence of 1mM ATP) a) that the ATPase activities of UM alone and of PM alone were both very low, and b) that both UM and PM sedimented at the same rate of approximately 10 S on ultracentrifugation. These observations are in support of our electron microscopic observation that high concentrations of MgCl₂ were required to observe ATP-resistant filaments of gizzard myosin, either UM or PM.
From these studies, it seems that the various differences so far reported between skeletal and gizzard contractile proteins arise from one basic difference, that high concentrations of magnesium are required for formation of ATP-resistant filaments from gizzard myosin whereas they are not required at all for that from skeletal myosin.
6. The turbidity of gizzard myosin suspensions underwent a reversible change on addition of myosin light-chain kinase (MLCK) with Ca²⁺ and on subsequent addition of myosin light-chain phosphatase (MLCP) with or without Ca²⁺. The reversible change in the turbidity (of myosin suspensions) was associated with that in the state of phosphorylation of myosin light chains, as well as with that in the assembly of myosin molecules which was examined by electron microscopy and by ultracentrifugation. These results provide further support for and are an extension of a previous proposal

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In 1978, we (Suzuki et al., J. Biochem. 84, 1529) reported the very interesting finding that in a medium of 0.2M KCl (and 10mM MgCl₂), addition of ATP induced a large increase in the sedimentation velocity of chicken gizzard myosin from approximately 6 S to 10 S. Moreover, our flow birefringence study suggested that 10 S-myosin was not much different from 6 S-myosin in the particle length. Therefore, we concluded that ATP induced dimerization of gizzard myosin monomers.
In the present study, we reinvestigated 6 S-myosin and 10 S-myosin by the sedimentation equilibrium method, and found that both myosins had the same molecular weight of approximately 500, 000. We also studied the angular dependence of the light scattering intensity, and found that addition of ATP caused a large change in the radius of gyration of gizzard myosin; the radius of gyration of 6 S-myosin was 545 Å whereas that of 10 S-myosin was only 146 Å. Accordingly, our previous conclusion had to be withdrawn. Instead, we now put forward a new conclusion that ATP induces a large change in the conformation of gizzard myosin monomers.
We added two new observations: (a) The ATP-induced change in the myosin conformation and the large decrease in the ATPase activity of myosin were both reversible upon increasing the KCl concentration from 0.2M to 0.3M. (b) The large decrease in the ATPase activity and the ATP-induced increase in the sedimentation velocity were also observed with gizzard heavy meromyosin when the KCl concentration decreased to lower than 0.3M.

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Ganglioside analogues (gangliosidoides) with an electron spin resonance label in a long aliphatic hydrocarbon chain were used to investigate the possible insertion of the sialoglycolipid into the plasma membrane of cells. Three types of ESR signals observed in the labeled glycolipids were distinguished. They characteristically indicate an isotropic tumbling motion of spin label in solution, the micellar state of the glycolipid, and an anisotropic motion in a lipid bilayer. Below CMC, gangliosidoide carrying one aliphatic hydrocarbon chain showed an isotropic tumbling motion. After the gangliosidoide had been incubated with liposomes or blood cells, there was an immediate change to an ESR signal showing an anisotropic motion. The signal was typical of the spin-label in liposomes prepared in the presence of spin-labeled sialoglycolipid. It can be concluded that the gangliosidoide was inserted into the lipid phase of liposomal or cellular membranes from the incubation medium.
The overall splitting (2A_//) of 5 SL-gangliosidoides in membranes was larger than those of 5 SL-galactosylceramide, 5 SL-phosphatidylcholine, and 5 SL-stearic acid, though the 2A_//of 12 SL-gangliosidoide was almost the same as those of other lipids having a nitroxide group in the 12-position of an acyl chain. This indicates that the head group movement is restricted in gangliosidoide molecules.

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A novel procedure of operational ease and reproducibility for the partial purification of DNA-dependent RNA polymerase [EC 2. 7. 7. 6] from Escherichia coli is reported. It utilizes liquid phase partitions with polyethylene glycol and Dextran, ammonium sulfate fractionations and chromatography on a QAE-Sephadex A-50 column.
A copurified protein in the partially purified preparation was isolated and identified as glutamine synthetase [L-glutamate: ammonia ligase (ADP) EC 6. 3. 1. 2].

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Superoxide anion production in peritoneal polymorphonuclear leukocytes obtained from guinea pigs was stimulated by in vitro treatment with membrane-perturbing agents, such as cytochalasin D, concanavalin A, phorbol myristate acetate, myristate, digitonin, and NaF. Vinblastine modified these stimulating effects on the superoxide anion production, but its modifying effect was not uniform. The effect of cytochalasin D was stimulated by vinblastine at the concentration of 10^-5-10^-7M, whereas it was inhibited at the concentration of 10^-4M. At 10^-4-10^-5M, vinblastine was inhibitory to the effect of concanavalin A, and lower concentrations had no significant effect. Stimulation of the superoxide anion production by phorbol myristate acetate and myristate was further enhanced by vinblastine at any concentration in the range of 10^-4-10^-8M with peaks at 10^-6 and 10^-5M, respectively. Vinblastine had little effect on the stimulation of the superoxide anion production by digitonin and NaF throughout the concentration range examined. The mechanism of the interaction of these membrane-perturbing stimulants and vinblastine is discussed.

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A new enzyme which hydrolyzes N-long chain acyl glutamic acid was found in cell-free extracts of Pseudomonas diminuta. Two active fractions (long acyl aminoacylase I and II) were separated by DEAE-cellulose column chromatography. The long acyl aminoacylase I was purified about 650-fold, and the purified preparation was electrophoretically homogeneous. The molecular weight was estimated to be 300, 000 by gel filtration. The enzyme was unique in its substrate specificity. It hydrolyzed only N-long acyl glutamic acid and could not react with other N-acyl amino acids. Lauroyl (C₁₂)-, myristoyl (C₁₄)-, and palmitoyl (C₁₆)- glutamic acid were good substrates, but acetyl glutamic acid was not hydrolyzed. Therefore this enzyme is considered to be a new acylase which is specific for N-long chain acyl glutamic acid, and it is designated as N-long acyl glutamic acid amidohydrolase [EC 3. 5. 1 group].

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The ketogenic effect of glucagon and its mechanism have been studied in isolated and cultured hepatocytes. The effects of _l-carnitine and insulin were also studied. Glucagon at 100 ng/ml consistently stimulated ketogenesis by 70-80% within 1 h with 0.3mM palmitate in hepatocytes isolated from rats on regular laboratory chow. Glucagon failed to exhibit a ketogenic effect, but inhibited the esterification of [U-¹⁴C] palmitate in the presence of (+) octanoyl-d-carnitine (0.5mM). (+) Octanoyl-d-carnitine inhibited the ketogenesis by 50%, but the inhibition was completely reversed by 3.3mM _l-carnitine. This indicates that the site of inhibition and the stimulatory step of ketogenesis by glucagon are at the carnitine palmitoyl-transferase (CPT) 1 reaction.
In mitochondria isolated from hepatocytes treated with glucagon for 30min, ketogenesis from added palmitate was increased by 50-55%. CPT 1 activity determined in these mitochondria was found to be increased by 25-50% with increased affinity for palmityl CoA over normal (K_m=13 μM versus 25 μM).
When cultured hepatocytes were supplemented with glucagon for 24 h, ketone body formation in the media was increased by 44%. In mitochondria isolated from these cells, the CPT level was elevated by 83%.
Insulin antagonized the ketogenic effect of a submaximal dose of glucagon in isolated hepatocytes, shifting the glucagon concentration which gives one-half maximal effect on ketogenesis from 5-7 ng/ml to 10-20 ng/ml. In cultured hepatocytes, insulin inhibited ketone body formation in the media and also significantly suppressed CPT activity by 34%. Although 1-carnitine enhanced ketogenesis in isolated hepatocytes, it had no effect in the cultured hepatocytes. These observations indicate that glucagon stimulates ketogenesis through early activation of CPT activity, possibly through cyclic AMP. Also, glucagon seems to induce the enzyme, while insulin suppresses CPT as a long-term regulation mechanism.

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Analysis of the quantitative precipitin reaction of adenylate kinase from porcine skeletal muscle with goat anti-adenylate kinase antiserum indicated that there are at least four antigenic determinants on the enzyme molecule. Porcine adenylate kinase was cleaved with cyanogen bromide, and four peptides were fractionated by ion-exchange chromatographies. Three fragments, CBb (2-56), CBfN (81-125), and CBfC (126-194), inhibited the quantitative precipitin reaction of intact adenylate kinase with goat antiserum. CBb, CBfN, and CBfC also inhibited the binding of ¹²⁵I-labeled adenylate kinase to the specific antibody purified from goat antiserum. In both inhibition studies, the inhibitory activity of each fragment was extremely high, and reached 70% or more in the latter case. From these results and in view of the presence of the sequence -Glu-Glu-X-X'-Lys (or Arg)-Lys- in each immunochemically active fragment, we suggest that these fragments have similar antigenic determinants which are cross-reactive.

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Rat liver mitochondria irradiated with external electric pulses (760 V/cm, 30 ms, rectangular) catalyzed net ATP synthesis of 0.055 nmol/mg protein/pulse. The amount of ATP synthesized increased on increasing the number, voltage and duration of the electric pulses. There was no apparent threshold voltage or duration for ATP synthesis in the ranges tested (0-760 V/cm and 0.05-30 ms). The energy applied to the mitochondrial membrane within a much shorter time than the turnover time of H⁺-ATPase must be stored in the membrane until it is utilized for ATP synthesis. Synthesis of ATP was inhibited by the specific H⁺-ATPase inhibitor aurovertin and by high concentrations of uncouplers. The energy transfer inhibitors oligomycin and dicyclohexylcarbodiimide had no effect on synthesis of ATP in mitochondria by electric pulses, but inhibited oxidative phosphorylation under the same conditions. In contrast to the pulse-driven ATP synthesis in subchloroplast particles, that in intact mitochondria required hexokinase-glucose, higher ADP concentration, lower osmolarity and protection against the pH effect of the electrodes.

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RecA protein is essential to general genetic recombination in Escherichia coli. In the presence of ATP, a stoichiometric amount of recA protein forms D-loops from superhelical closed-circular DNA (form I DNA) and homologous single-stranded fragments, and subsequently dissociates the D-loops. Under appropriate conditions, the hydrolysis of ATP by recA protein depends on the presence of both doublestranded DNA and homologous single-stranded fragments (homology-dependent hydrolysis). In the presence of form I DNA, most of the homology-dependent hydrolysis of ATP by recA protein is related to the dissociation of D-loops rather than the formation of D-loops. RecA protein also promoted the homology-dependent hydrolysis of ATP in the presence of nicked-circular DNA (form II DNA), but unlike the case of form I DNA, this hydrolysis was associated with an increase in the amount of mature D-loops that were detected by the D-loop assay.
When double-stranded DNA was superhelical, the homology-dependent hydrolysis of ATP continued at the same rate even after all the D-loops were dissociated. This correlates with our earlier observation that in the process of formation and dissociation of D-loops, form I DNA was converted to an inactive substrate without any apparent damage to the DNA, probably by the formation of a complex with recA protein.
All of the observations described above can be explained by a model in which a common mechanism causes dissociation of D-loops from form I DNA, inactivation of form I DNA, and growth of D-loops in form II DNA. The mechanism might involve cooperative binding of recA protein to the duplex DNA from the site of the nascent D-loop, resulting in unidirectional unwinding of the duplex DNA.

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The chemical reaction of p-bromophenacyl bromide (BPB) with His 48 of cobra venom phospholipases A₂ (N. naja siamensis, N. naja kaouthia, and N. naja atra) was studied at 25&C and ionic strength 0.1 by following the decreases in the fluorescence intensity of 8-anilino-l-naphthalene sulfonate (ANS) and/or in the enzymatic activity.
The effect of the BPB concentration on the pseudo-first-order rate constant, _??_obs, was studied for the N. naja atra enzyme and the dissociation constants of a noncovalent intermediate were determined to be 5.6×10^-4 and 1.8×10^-4M at pH 8.4 and 9.3, respectively.
The pH-dependence curve of _??_obs, obtained at fixed concentration (s) of BPB was found to be biphasic for all three enzymes. The analysis showed that two ionizable groups with pK values of 7.3 and 8.55 participated in this reaction. The reaction of BPB with the modified enzyme of N. naja atra lacking the N-terminal octapeptide, which had been prepared by the CNBr-cleavage method, was followed by measuring the decrease in the tryptophyl fluorescence. Since this modified enzyme showed a monophasic pH-dependence curve lacking the latter transition, the former group was assigned to His 48 and the latter to the α-amino group located nearby in the active site. The pK value of His 48 determined at present was in good agreement with that estimated from the pH dependence of the binding constant of Ca²⁺ (Teshima et al. (1981) J. Biochem. 89, 13-20). The pK value of the α-amino group of this enzyme, 8.55, was found to be somewhat higher than that of the A. halys blomhoffii enzyme, 7.3 (Ikeda & Samejima (1981) J. Biochem. 90, 799-804 and Ikeda et al. (1981) J. Biochem. 90, 1125-1130), but quite similar to that for the porcine pancreatic enzyme, 8.4 (van Dam-Mieras et al. (1976) Nobel Symp. 34, 177-197 and Slotboom et al. (1978) Biochemistry 17, 4593-4600).

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The aerobic oxidation of rifamycin SV to rifamycin S was catalyzed by horseradish peroxidase. The oxidation rate increased with increasing pH. The oxidation was inhibited by catalase, but superoxide dismutase enhanced the oxidation rate of rifamycin SV. Superoxide inhibited the oxidation and superoxide dismutase relieved the inhibition of oxidation by superoxide. An oxidation mechanism of rifamycin SV catalyzed by horseradish peroxidase which accounts for these data is proposed and a convenient positive assay for superoxide dismutase, using the rifamycin SV-horseradish peroxidase-O₂ system, is described.

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1. Clam-foot paramyosin strongly inhibited superprecipitation of clam-foot actomyosin whereas it did not at all affect its ATPase reaction. On the other hand, superprecipitation of rabbit actomyosin and its ATPase reaction were both inhibited by clam foot paramyosin.
2. Clam-foot paramyosin did not at all affect the Mg-ATPase activity of clam-foot myosin alone (with no actin) but inhibited that of rabbit skeletal myosin alone.
3. Clam-foot paramyosin did not affect the ATPase reaction of rabbit acto-clam myosin but inhibited its superprecipitation. In other words, the effect of paramyosin on the ATPase reaction of actomyosin was simply dependent on the myosin source.
4. Clam-foot tropomyosin slightly enhanced superprecipitation of clam-foot actomyosin and scarcely affected its ATPase reaction, but it inhibited the reactions both of rabbit skeletal actomyosin and of rabbit acto-clam myosin.
5. Rabbit skeletal light-meromyosin (LMM) inhibited the ATPase and superprecipitation reactions of rabbit skeletal actomyosin, but it scarcely affected the ATPase reaction of rabbit acto-clam myosin while inhibiting its superprecipitation. In other words, rabbit LMM behaved the same way as clam paramyosin did.
6. Speculating on these findings and our previous findings, we suggest that the formation of a certain filamentous structure is the essential link between the ATPase reaction and contraction in molluscan muscles.

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In an attempt to clarify the mechanism of cardiac neuropathy in diabetes mellitus, Wister male rats were made diabetic by streptozotocin injection for 11 to 13 weeks, and catecholamine concentrations of hearts were determined by high performance liquid chromatography. No apparent histological changes were found in hearts and kidneys of any group of rats. Controls used were age-matched normal rats and Goldblatt-hypertensive rats, because streptozotocin induced diabetic rats appeared to be significantly hypertensive. Heart norepinephrine concentrations of diabetic rats and diabetic-Goldblatt-hypertensive rats were markedly higher (8, 380±300 pmol/g tissue and 6, 980±390, respectively) compared with those of controls and Goldblatt-hypertensive rats (2, 700±470 and 2, 010±300, respectively). These results suggest some disturbances in catecholamine secretion in diabetic hearts before typical microangiopathic changes take place.

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1. The actin-activated Mg²⁺-ATPase activity of gizzard HMM increased in proportion to the square of the extent of LC phosphorylation. This result indicates that the LCs of HMM are randomly phosphorylated, and the phosphorylation of both heads of HMM is required for the activation of HMM Mg²⁺-ATPase by F-actin.
2. In 75mM KCl, the Mg²⁺-ATPase activity of gizzard myosin was activated by F-actin only slightly when a half of the total LC was phosphorylated. From 1 to
2 mol LC phosphorylation, the activity was enhanced by F-actin almost linearly. In 30mM KCl, the activity of acto-gizzard myosin increased sigmoidally with increase in the extent of LC phosphorylation. On electron microscopy, side-by-side aggregates of myosin filaments were observed in 30mM KCl, but not in 75mM KCl. It was suggested that the activation of the Mg²⁺-ATPase activity of acto-gizzard myosin LC phosphorylation is modified by formation of myosin filaments and their aggregates.
3. The relationship between the actin-activated Mg²⁺-ATPase activity of HMM or myosin and the extent of LC phosphorylation was unaffected by tropomyosin.

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A simple method for the purification of antiglycolipid antibody is described. Polystyrene latex beads coated with gangliotetraosylceramide (GA₁) were used as immunoadsorbent for the purification of anti-GA₁ antibody. The antibody bound to the GA₁-coated latex beads was released with 1M NaSCN solution. The glycolipidcoated latex beads seem valuable not only for the purification of antibodies but also as a tool for investigating the function of glycolipids.

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The limited tryptic digestion of the heavy chain of chymotryptic myosin sugfragment-1 gives three major fragments (50 K, 27 K, and 20 K daltons) without much additional proteolysis. The distribution of thiols over the tryptic fragments of the heavy chain was estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. Contents of thiol in the 50 K, 27 K, and 20 K fragments were found to be 4, 1, and 4, respectively.

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Amino acid compositions of 356 proteins are expressed as points in an 18 dimensional space of 18 axes representing the contents of amino acids. The proteins are classified into four groups of intra- and extracellular enzymes and nonenzymes according to analysis of the distribution of the points. The groups have a significant correlation to four folding types of secondary structures, and extra- and intracellular proteins to those with and without disulfide bond (s), respectively. The location and function of a protein seem to determine its amino acid composition and folding type.

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In order to elucidate how phosphatidylserine enhances histamine release induced by concanavalin A from rat mast cells, two phosphatidylserine analogues were synthesized and their effects were examined. A dialkyl analogue of phosphatidylserine, both ester bonds of which are replaced by ether bonds, potentiated histamine secretion from mast cells stimulated by concanavalin A, although it was much less active than natural phosphatidylserine. An alkyl analogue of phosphatidylserine, in which an ether bond is present at the 1 position, showed almost the same low activity as the dialkyl analogue. It is suggested that deacylation of the incorporated phosphatidylserine by phospholipase A is not required for potentiation by phosphatidylserine and the existence of an ester bond at position 1 is important for phosphatidylserine to enhance concanavalin A-induced histamine secretion from mast cells.

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